Gene therapy for restenosis using an adenoviral vector

ABSTRACT

A method for treating restenosis by gene therapy is disclosed, said method comprising delivering a recombinant suicide-gene-containing adenovirus.

[0001] The present invention relates to a method for treatment ofrestenosis by gene therapy, comprising the administration of arecombinant adenovirus containing a suicide gene. It also relates toparticular pharmaceutical compositions permitting the local andeffective administration of recombinant viruses.

[0002] Atherosclerosis is a complex, polygenic disease which is definedin histological terms by deposits (lipid or fibrolipid plaques) oflipids and of other blood derivatives in the wall of the large arteries(aorta, coronary arteries, carotid). These plaques, which are more orless calcified according to the degree of progression of the process,may be coupled with lesions and are associated with the accumulation inthe arteries of fatty deposits consisting essentially of cholesterolesters. These plaques are accompanied by a thickening of the arterialwall, with hypertrophy of the smooth muscle, appearance of foam cellsand accumulation of fibrous tissue. The atheromatous plaque protrudesmarkedly from the wall, endowing it with a stenosing characterresponsible for vascular occlusions by atheroma, thrombosis or embolismwhich occur in those patients who are most affected. These lesions canhence lead to very serious cardiovascular pathologies such asinfarction, sudden death, cardiac insufficiency, stroke, and the like.

[0003] Since 1977, the technique of angioplasty has been developed topermit a non-surgical intervention in respect of the atherosclerosisplaque. However, the treatment of an atherosclerotic lesion byangioplasty results very frequently (up to 50% of cases in some studies)in a restenosis following mechanical injury of the arterial wall. A keyevent in this mechanism is the proliferation and migration of vascularsmooth muscle cells (VSMC) from the media to the intima, in particularas a result of the absence of protection and/or feedback controlexercised by the endothelial cells of the intima.

[0004] The treatment of restenosis by administration of chemical orproteinaceous substances capable of killing vascular smooth muscle cellshas been proposed in the prior art. Thus, psolaren derivatives,incorporated by proliferative cells and then sensitizing these cells tothe action of light, have been used (March et al., 1993, circulation,87:184191). Similarly, some cytotoxins consisting of a fusion proteinbetween a plant or bacterial toxin fragment and a growth factor havealso been used (Pickering et al., J. Clin. Invest., 1993, 91:724-729;Biro et al., 1992, Circ. Res., 71:640-645; Casscells et al., Proc. Natl.Acad. Sci. USA, 1992, 89:7159-7163). However, these treatments have manydrawbacks, such as their low specificity, their indifferent efficacy, aconsiderable delay in acting and a potential toxicity.

[0005] The present invention offers an advantageous approach to thisproblem. The present invention provides, in effect, an especiallyeffective and selective method for the treatment of postangioplastyrestenosis by gene therapy. The method of the present invention consistsmainly in administering a recombinant adenovirus containing a suicidegene, capable of specifically sensitizing proliferating vascular smoothmuscle cells to a therapeutic agent. Simultaneous or subsequentadministration of this therapeutic agent then brings about the selectivedeath of the sensitized cells.

[0006] The advantages of the present invention lie, in particular, inthe high capacity of the adenoviruses of the invention to infectproliferating vascular smooth muscle cells. This enables relativelysmall amounts of active principle (recombinant adenovirus) to be used,and also permits an effective and very rapid action on the sites to betreated. The adenoviruses of the invention are also capable ofexpressing at very high levels the suicide genes introduced, therebyendowing them with a very effective therapeutic action. Furthermore, onaccount of their episomal character, the adenoviruses of the inventionhave a limited persistence in proliferative cells, and hence a transienteffect entirely suited to the desired therapeutic effect. Lastly, theApplicant has also developed an especially advantageous method ofadministration, which enables certain target cells essential to thedesired therapeutic effect to be infected with great efficacy.

[0007] A first subject of the invention hence relates to the use of adefective recombinant adenovirus containing a suicide gene for thepreparation of a pharmaceutical composition intended for the treatmentof restenosis.

[0008] As mentioned above, for the purposes of the present invention,suicide gene is understood to mean any gene whose expression productendows the infected cell with a sensitivity to a therapeutic agent. Asan example, there may be mentioned the thymidine kinase gene, whoseexpression product endows mammalian cells with a sensitivity to certaintherapeutic agents such as ganciclovir or acyclovir, or the cytosinedeaminase gene, whose expression product endows mamalian cells with asensitivity to 5-fluorocytosine (5-FC).

[0009] Herpes simplex virus thymidine kinase is capable ofphosphorylating nucleoside analogues such as acyclovir and ganciclovir.These modified molecules may be incorporated in a DNA chain undergoingelongation, which results in the cessation of DNA synthesis and bringsabout the death of the cell (F. L. Moolten, Cancer Res. 46 (1986) 5276).This strategy thus enables cells expressing the TK gene to bespecifically eliminated. Furthermore, since the DNA synthesis is thetarget of the toxicity, only cells undergoing division are affected.

[0010] More preferably, the human herpesvirus thymidine kinase (hHSV-1TK) gene is used in the context of the present invention. The sequenceof this gene has been described in the literature (see, in particular,McKnight et al., Nucleic Acid. Res. 8 (1980) 5931). It is also possibleto use derivatives of this sequence displaying greater substratespecificity or better kinase activity. Such derivatives may, inparticular, be obtained by mutagenesis at the binding site, as describedpreviously (Balasubramaniam et al., J. Gen. Virol. 71 (1990) 2979; Muniret al., JBC 267 (1992) 6584).

[0011] It is also possible to use the cytosine deaminase gene, whoseexpression product endows mammalian cells with a sensitivity to5-fluorocytosine (5-FC). Cytosine deaminase is capable of catalyzing thedeamination of cytosine to uracil. Cells which express this gene arehence capable of converting 5-fluorocytosine (5-FC) to 5-fluorouracil(5-FU), which is a toxic metabolite. The sequence of this gene has beendescribed in the literature (Anderson et al., Arch. Microbiol. 152(1989) 115).

[0012] More generally, any gene capable of endowing infected cells witha sensitivity to a therapeutic agent may be used in the context of thepresent invention. The thymidine kinase gene constitutes an especiallyadvantageous embodiment.

[0013] For the construction of the adenoviruses according to theinvention, different serotypes may be used. There are, in effect, manyserotypes of adenovirus, whose structure and properties vary somewhat.Among these serotypes, it is preferable however to use, in the contextof the present invention, human adenoviruses type 2 or 5 (Ad 2 or Ad 5)or adenoviruses of animal origin (see Application FR 93/05954). Amongadenoviruses of animal origin which are usable in the context of thepresent invention, adenoviruses of canine, bovine, murine (for example:Mav1, Beard et al., Virology 75 (1990) 81), ovine, porcine, avian oralternatively simian (for example: SAV) origin may be mentioned.Preferably, the adenovirus of animal origin is a canine adenovirus, morepreferably a CAV2 adenovirus [Manhattan or A26/61 (ATCC VR-800) strain,for example]. It is preferable to use adenoviruses of human or canine ormixed origin in the context of the invention.

[0014] As stated above, the adenoviruses according to the invention aredefective, that is to say they are incapable of replicating autonomouslyin the target cell. Generally, the genome of the defective adenovirusesused the context of the present invention hence lacks at least thesequences needed for replication of the said virus in the infected cell.These regions may be either removed (in their entirety or partially), orrendered non-functional, or replaced by other sequences, and inparticular by the suicide gene. Preferably, the defective adenovirusnevertheless retains the sequences of its genome which are needed forencapsidation of the viral particles.

[0015] Preferably, the defective adenoviruses of the invention comprisethe ITRs, a sequence permitting encapsidation and the suicide gene.Still more preferably, in the genome of the adenoviruses of theinvention, the E1 gene and at least one of the genes E2, E4, L1-L5 arenon-functional. The viral gene of interest may be renderednon-functional by any technique known to a person skilled in the art,and in particular by total elimination, substitution, partial deletionor addition of one or more bases in the gene or genes of interest. Suchmodifications may be obtained in vitro (on the isolated DNA) or in situ,for example by means of genetic engineering techniques, or alternativelyby treatment using mutagenic agents.

[0016] More preferably, a defective adenovirus rendered non-functionalby a total or partial deletion of the E1 region and a deletion in the E4region is used. The E4 region comprises 7 reading frames. The deletionin the E4 region may be transcomplemented by the presence, in the cellline used for multiplication of the viruses, either simply of thereading frame ORF6, or of the reading frames ORF6 and ORF6/7.

[0017] Preferred adenoviruses according to the invention are chosen fromthe following:

[0018] Defective recombinant adenovirus ΔE1, ΔE4 comprising a deletionof all or part of the E1 region and a deletion of all or part of the E4region.

[0019] Defective recombinant adenovirus ΔE1, ORF3⁻, ORF6⁻ comprising adeletion of all or part of the E1 region and of nucleotides 34801-34329and 34115-33126 of the E4 region.

[0020] Defective recombinant adenovirus ΔE1, ΔE4, ORF1⁺ comprising adeletion of all or part of the E1 region and a deletion of the E4 regionexcept for the reading frame ORF1. More specifically, the deletion inthe E4 region has its 5′ end included in the reading frame ORF7 and its3′ end included in the reading frame ORF2. For example, in the regioncovering nucleotides 33093-35053.

[0021] Defective recombinant adenovirus ΔE1, AE4, ORF4⁺ comprising adeletion of all or part of the E1 region and a deletion of the E4 regionexcept for the reading frame ORF4. More especially, two deletions arecarried out, one whose 5′ end is included in the reading frame ORF7 andwhose 3′ end is located in the reading frame ORF6, the other whose 5′end is included in the reading frame ORF3 and whose 3′ end is located inthe reading frame ORF1 or in the promoter region of E4. For example, adeletion covering nucleotides 33093-33695 and a deletion coveringnucleotides 34634-35355.

[0022] Defective recombinant adenovirus ΔE1, ΔE4 comprising a deletionof all or part of the E1 region and a deletion covering the whole of theE4 region, chosen, for example, from the following deletions:nucleotides 32720-35835, or 33466-35355, or 33093-35355.

[0023] The construction of these vectors is described in Patents No. FR9500749 and No. FR 9506532.

[0024] The defective recombinant adenoviruses according to the inventionmay be prepared by any technique known to a person skilled in the art(Levrero et al., Gene 101 (1991) 195, EP 185,573; Graham, EMBO J. 3(1984) 2917). In particular, they may be prepared by homologousrecombination between an adenovirus and a plasmid carrying, inter alia,the suicide gene. Homologous recombination takes place aftercotransfection of the said adenovirus and said plasmid into a suitablecell line. The cell line used should preferably (i) be transformable bythe said elements, and (ii) contain the sequences capable ofcomplementing the portion of the genome of the defective adenovirus,preferably in integrated form in order to avoid risks of recombination.As an example of a line, there may be mentioned the human embryonickidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) whichcontains, in particular, integrated in its genome, the left-hand portionof the genome of an Ad5 adenovirus (12%). Strategies of construction ofvectors derived from adenoviruses have also been described inApplications Nos. FR 93/05954 and FR 93/08596.

[0025] Thereafter, the adenoviruses which have multiplied are recoveredand purified according to standard techniques of molecular biology, asillustrated in the examples.

[0026] Advantageously, in the adenoviruses of the invention, the suicidegene is placed under the control of a promoter permitting its expressionin infected cells. This promoter can be the one belonging to the suicidegene, a heterologous promoter or a synthetic promoter. In particular,promoters originating from eukaryotic or viral genes may be used. Forexample, it is possible to use promoter sequences originating from thegenome of the cell which it is desired to infect. Similarly, thepromoter sequences may originate from the genome of a virus, includingthat of the virus used. In this connection, the E1A, MLP, CMV, RSV LTR,and the like, promoters may, for example be mentioned. In addition,these expression sequences may be modified by adding activation orregulatory sequences or sequences permitting a tissue-specificexpression. It can, in effect, be especially advantageous to useexpression signals which are active specifically or predominantly invascular smooth muscle cells, so that the suicide gene is expressed andproduces its effect only when the virus has actually infected a vascularsmooth muscle cell. Among promoters which are active specifically orpredominantly in vascular smooth muscle cells, the promoter of α-actinof smooth muscle may be mentioned in particular.

[0027] In a particular embodiment of the invention, a defectiverecombinant adenovirus is used which comprises a suicide gene under thecontrol of a viral promoter, preferably chosen from the RSV LTR and theCMV early promoter.

[0028] According to another advantageous embodiment, the promoter usedis one which is active specifically or predominantly in vascular smoothmuscle cells.

[0029] The present invention thus provides an especially effectivemethod for the treatment of restenosis. Moreover, to increase furtherthe efficacy and specificity of the treatment, the Applicant hasdeveloped a method permitting a local administration of the recombinantadenoviruses at the sites to be treated. More especially, this method isbased on the use of an angioplasty balloon coated with a hydrophilicfilm (for example a hydrogel) impregnated with adenovirus, which maythus be applied precisely to the site to be treated and permit a localand effective release of the adenoviruses at the cells to be treated.

[0030] In addition, the Applicant showed that, on healthy arteries, thismethod of administration made it possible to infect a high percentage ofcells of the media (up to 9.6%), which are the most logical targets forthe prevention of restenosis.

[0031] In a most particularly advantageous aspect, the Applicant alsoshowed that the virus and the method of the invention permitted aneffective and selective transfer of genes into an atheromatous artery.More especially, the Applicant has demonstrated for the first time thecapacity of adenoviruses to transfer a therapeutically effective geneinto an atheromatous artery. This is absolutely essential, since thetherapeutic efficacy of the treatment of restenosis is conditional on ademonstration of the capacity to transfer the therapeutic gene, into thecorrect cells and with suitable efficacy, under the physiopathologicalconditions. Atheromatous arteries are characterized by the presence inthe intima (i) of deposits of extracellular matrix, (ii) of lipiddeposits consisting essentially of foam cells of the macrophage type and(iii) of proliferating smooth muscle cells.

[0032] The results presented below show that, in these atheromatousarteries, the viruses according to the invention permit a lowerpercentage of infection but which is of greater specificity (takingaccount, in fact, of the presence of macrophage type cells in this case,macrophage cells not being transduced) and which is accompanied by asubstantial therapeutic efficacy. The results obtained show, inparticular, a very selective transfer of the adenovirus into the targetcells, that is to say the proliferating smooth muscle cells. Out of thewhole cell population present in the atheromatous zone, more than 95% ofthe infected cells are vascular smooth muscle cells. Thus, themacrophage cells present in the intima are not infected at all (noinfected macrophage cell was detected). As regards the proliferatingsmooth muscle cells (in the neointima), the treatment according to theinvention enables a percentage of less than 1% (for example 0.2%) to beinfected. This is much less than the results described previously inhealthy arteries or those possessing lesions of the wall but which donot represent a physiopatholigical situation of restenosis (endothelialabrasion of a healthy artery). The Applicant also showed that theinfection of this small percentage of cells nevertheless permitted asubstantial therapeutic effect, demonstrated, in particular, bymeasurement of the luminal diameter. This result is especiallysurprising and implies the existence of an induced cytotoxic effect(“bystander” effect) in vivo. Hence the invention describes for thefirst time a method permitting the selective transfer of genes intoproliferating vascular smooth muscle cells in an atheromatous artery,comprising the administration into the said artery of a defectiverecombinant adenovirus containing the said gene by means of anangioplasty balloon catheter. The term selective transfer implies atransfer essentially into the proliferating vascular smooth muscle cellsand no transfer into the surrounding macrophage cells. This methodpermits a treatment of restenosis by transfer of a suicide gene such asthe TK gene, followed by treatment with ganciclovir or acyclovir, forexample. This method of treatment is, in addition, characterized by aneffect of toxicity induced in vivo.

[0033] Another subject of the present invention relates to apharmaceutical composition comprising a defective recombinant adenovirusand a hydrogel. More specifically, the invention relates to acomposition comprising a defective recombinant adenovirus containing asuicide gene, and a hydrogel. The hydrogel used in the context of thepresent invention may be prepared from any biocompatible andnon-cytotoxic (homo- or co-)polymer. Such polymers have, for example,been described in Application W093/08845. Some of them, such as, inparticular, those obtained from ethylene oxide and/or propylene oxide,are commercially available.

[0034] The method of treatment of the invention hence advantageouslyconsists in introducing, at the site to be treated, a compositioncomprising a hydrogel impregnated with recombinant adenoviruses. Thehydrogel may be deposited directly on the surface of the tissue to betreated, for example during a surgical intervention. Advantageously, thehydrogel [lacuna] be introduced into the site to be treated by means ofa catheter, for example a balloon catheter, in particular duringangioplasty, thereby enabling any additional trauma due to a furtherintervention at the angioplasty site to be avoided. It is especiallyadvantageous for the impregnated hydrogel to be introduced into the siteto be treated by means of a balloon catheter protected by a sleeve. Asdescribed in the examples, the hydrogel possesses a large number ofadvantages: it enables sliding of the balloon to be improved, therebyenabling it to pass through heavily stenosed arteries. Furthermore, thehydrogel can be used with any type of angioplasty balloon, which enablesperfusion balloons to be used in particular. Thus, according to aparticular embodiment, the adenoviruses according to the invention areadministered by means of perfusion balloons, especially channelledballoon catheters (“channelled balloon angioplasty catheter”, MansfieldMecical, Boston Scientific Corp., Watertown, Mass.). The latter consistsof a conventional balloon covered with a layer of 24 perforated channelswhich are perfused via an independent lumen through an additionalinfusion orifice. These perfusion balloons, which make it possible tomaintain a blood flow and thus to decrease the risks of ischaemia of themyocardium, on inflation of the balloon, also enable a medicinal productto be delivered locally at normal pressure for a relatively long time,more than twenty minutes, which is necessary for an optimal infection.

[0035] It is especially advantageous to use a perfusion balloon cathetercoated with hydrogel. In this case, the advantages of both, that is tosay the possibility of keeping the balloon inflated for a longer periodof time by retaining the properties of facilitated sliding and ofsite-specificity of the hydrogel, are gained simultaneously. In thiscase, an optimal efficacy of infection is obtained.

[0036] The results presented in the examples demonstrate, in fact, theefficacy of this system for the percutaneous transfer of genes into thearterial walls.

[0037] Another subject of the present invention relates to apharmaceutical composition comprising a defective recombinant adenovirusand poloxamer. More specifically, the invention relates to a compositioncomprising a defective recombinant adenovirus containing a suicide gene,and poloxamer. Poloxamer 407 is a non-toxic, biocompatible polyol; it iscommercially available (BASF, Parsippany, N.J.).

[0038] Hence a method of treatment of the invention advantageouslyconsists in introducing, into the site to be treated, a compositioncomprising poloxamer impregnated with recombinant adenoviruses. Thepoloxamer may be deposited directly on the surface of the tissue to betreated, for example during a surgical intervention. Advantageously, thepoloxamer may be introduced into the site to be treated by means of acatheter, for example a balloon catheter, in particular duringangioplasty, thereby enabling any additional trauma due to a furtherintervention at the angioplasty site to be avoided. It is especiallyadvantageous for the impregnated poloxamer to be introduced into thesite to be treated by means of a balloon catheter protected by a sleeve.Poloxamer possesses essentially the same advantages as hydrogel whilehaving a lower viscosity.

[0039] It is especially advantageous to use a perfusion balloon cathetercoated with poloxamer, especially channelled balloon catheters. In thiscase, the advantages of both, that is to say the possibility of keepingthe balloon inflated for a longer period of time while retaining theproperties of facilitated sliding and of site-specificity of thepoloxamer, are gained simultaneously. In this case also, an optimalefficacy of infection is obtained.

[0040] The present invention will be described more completely by meansof the examples which follow, which are to be considered to beillustrative and non-limiting.

LEGEND TO THE FIGURES

[0041]FIG. 1: Diagram of the vector pONT-tk

[0042]FIG. 2: Diagram of the vector pRSV-tk

[0043]FIG. 3: Cytotoxic effect of the ganciclovir/Ad-LTR-tk combinationon smooth muscle cells in culture.

[0044]FIG. 4: Reduction of restenosis by adenoviral transfer of the tkgene and administration of ganciclovir.

GENERAL TECHNIQUES OF MOLECULAR BIOLOGY

[0045] The methods traditionally used in molecular biology, such aspreparative extractions of plasmid DNA, centrifugation of plasmid DNA ina caesium chloride gradient, agarose or acrylamide gel electrophoresis,purification of DNA fragments by electroelution, phenol orphenol/chloroform extraction of proteins, ethanol or isopropanolprecipitation of DNA in a saline medium, transformation in Escherichiacoli, and the like, are well known to a person skilled in the art andare amply described in the literature [Maniatis T. et al., “MolecularCloning, a Laboratory Manual”, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., (1982); Ausubel F.M. et al. (eds), “CurrentProtocols in Molecular Biology”, John Wiley & Sons, New York, (1987)].

[0046] Plasmids of the pBR322 and pUC type and phages of the M13 seriesare of commercial origin (Bethesda Research Laboratories).

[0047] To carry out ligation, the DNA fragments may be separatedaccording to their size by agarose or acrylamide gel electrophoresis,extracted with phenol or with a phenol/chloroform mixture, precipitatedwith ethanol and then incubated in the presence of phage T4 DNA ligase(Biolabs) according to the supplier's recommendations.

[0048] The filling in of 5′ protruding ends may be performed with theKlenow fragment of E.coli DNA polymerase I (Biolabs) according to thesupplier's specifications. The destruction of 3′ protruding ends isperformed in the presence of phage T4 DNA polymerase (Biolabs) usedaccording to the manufacturer's recommendations. The destruction of 5′protruding ends is performed by a controlled treatment with S1 nuclease.

[0049] In vitro site-directed mutagenesis using syntheticoligodeoxynucleotides may be performed according to the method developedby Taylor et al. [Nucleic Acids Res. 13 (1985) 8749-8764] using the kitdistributed by Amersham.

[0050] The enzymatic amplification of DNA fragments by the so-called PCR[Polymerase-catalyzed Chain Reaction, Saiki R. K. et al., Science 230(1985) 1350-1354; Mullis K. B. and Faloona F. A., Meth. Enzym. 155(1987) 335-350] technique may be performed using a “DNA thermal cycler”(Perkin Elmer Cetus) according to the manufacturer's specifications.

[0051] The verification of the nucleotide sequences may be performed bythe method developed by Sanger et al. [Proc. Natl. Acad. Sci. USA, 74(1977) 5463-5467] using the kit distributed by Amersham.

EXAMPLES Example 1 Construction of the Vector Ad-LTR-TK Carrying the TKGene Under the Control of the Rous Sarcoma Virus LTR (RSV LTR) Promoter(FIG. 1)

[0052] This example describes the construction of a recombinantadenovirus comprising the herpes simplex virus thymidine kinase (tk)gene under the control of a viral promoter (RSV LTR promoter). Thisadenovirus was constructed by homologous recombination between thedefective adenovirus Ad-dl1324 and plasmid pRSVtk carrying the tk geneunder the control of the RSV promoter (Example 1.3.). This plasmid wasconstructed from plasmid pONTtk (Example 1.1.) by substituting the RSVpromoter for the promoter which can be transactivated by EBNA1 (Example1.2.).

[0053] 1.1. Construction of plasmid pONTtk

[0054] a) Construction of plasmid p7tk1

[0055] This example describes the construction of plasmid p7tk1containing the 1131-base pair open reading frame of the tk gene (ATG114-116 and stop codon TGA 1242-1244), inserted into a multicloningsite.

[0056] The BglII-NcoI fragment containing the herpes simplex type 1virus thymidine kinase (tk) gene was isolated from plasmid pHSV-106(marketed by Gibco BRL), repaired by the action of the Klenow fragmentand then inserted at the SmaI site of plasmid pGEM7zf(+) (marketed byPromega). The SmaI and BgIII sites are destroyed during this step, theNcoI site is preserved.

[0057] The plasmid obtained was designated p7tkl.

[0058] b) Construction of plasmid pONT1.

[0059] This example describes the construction of a plasmid containing achimeric promoter consisting of a sequence needed for transactivation bythe antigen EBNA1 and of the EBV virus TP1 promoter.

[0060] The EcoRI(7315)-SmaI(8191) fragment of the EBV virus was isolatedfrom the strain B95-8. The complete sequence of the EBV virus has beendescribed by Baer et al. (Nature 310 (1984) 207). This fragment containsthe sequences needed for transactivation by the nuclear antigen 1(EBNA1) (D. Reisman & B. Sugden, 1986, Molecular and Cellular Biology,vol. 6 pp. 3838-3846). This fragment was then fused to the NruI(166241)-PstI(166 559) fragment of EBV B95-8 (the PstI site was digestedwith T4 polymerase), containing the TP1 promoter. The chimeric promoterthereby obtained was then inserted into the multicloning site of theplasmid pBluescript II SK to generate plasmid pONT1.

[0061] c) Construction of plasmid pONTtk

[0062] Plasmid pONTtk contains the herpes simplex virus thymidine kinase(tk) gene cloned into plasmid p7tk1, under the control of the chimericpromoter EBNA1-RE/TP1 cloned into plasmid pONT1.

[0063] To construct this plasmid, the BamHI-XhoI fragment of pONT1,which contains the chimeric promoter transactivated by EBNA-1 andEBNA-2, and the XhoI-ClaI fragment of p7tkl, which contains the openreading frame of tk, were cloned at the BamHI (478) and ClaI (4550)sites of plasmid pAd.RSVbgal. Plasmid pAd.RSVbGal contains, in the 5′→3′orientation,

[0064] the PvuII fragment corresponding to the left-hand end of the Ad5adenovirus comprising: the ITR sequence, the origin of replication, theencapsidation signals and the E1A enhancer;

[0065] the gene coding for b-galactosidase under the control of the RSV(Rous sarcoma virus) promoter,

[0066] a second fragment of the Ad5 adenovirus genome, which permitshomologous recombination between plasmid pAd.RSVbGal and the adenovirusd1324. Plasmid pAd.RSVbGal has been described by Stratford-Perricaudetet al., (J. Clin. Invest. 90 (1992) 626).

[0067] All the cloning sites are preserved. The plasmid obtained wasdesignated.pONTtk.

[0068] 1.2. Construction of plasmid pRSVtk

[0069] This plasmid was constructed from plasmid poNTtk (Example 1.1.)by substituting the RSV promoter for the promoter which can betransactivated by EBNA1. To this end, the RSV promoter was isolated inthe form of a BamHI-SalI fragment from plasmid pAd.RSV.βgal(Stratford-Perricaudet et al., J. Clin. Invest. 90 (1992) 626), and thencloned at the BamHI(478) and SalI(1700) sites of plasmid pONTtk. Theresulting plasmid was designated pRSVtk (FIG. 1).

[0070] 1.3. Construction of the recombinant adenovirus Ad-RSV-tk

[0071] The vector pRSVtk was linearized and cotransfected with adefective adenoviral vector into helper cells (line 293), providing intrans the functions encoded by the adenovirus E1 (E1A and E11B) regions.

[0072] More precisely, the adenovirus Ad-RSV-tk was obtained by in vivohomologous recombination between the mutant adenovirus Ad-dl1324(Thimmappaya et al., Cell 31 (1982) 543) and the vector pRSVtk,according to the following protocol: plasmid pRSVtk, linearized withXmnI, and the adenovirus Ad-dl1324, linearized with the enzyme ClaI,were cotransfected into line 293 in the presence of calcium phosphate,to permit homologous recombination. The recombinant adenoviruses thusgenerated were selected by plaque purification. After isolation, therecombinant adenovirus DNA was amplified in cell line 293, leading to aculture supernatant containing the unpurified defective recombinantadenovirus having a titre of approximately 10¹⁰ pfu/ml.

[0073] The viral particles were then purified by centrifugation on acaesium chloride gradient according to known techniques (see, inparticular, Graham et al., Virology 52 (1973) 456). The adenovirusAd-RSV-tk may be stored at −80° C. in 20% glycerol.

Example 2 Activity of an Adenovirus According to the Invention in thePresence of Ganciclovir on Smooth Muscle Cells in Culture

[0074] The activity of the adenovirus containing the TK gene, preparedin Example 1, was checked on in vitro models of smooth muscle cells. Tothis end, smooth muscle cells isolated from rat and rabbit aorta wereinfected with the recombinant adenovirus Ad-RSV-tk and incubated in thepresence of ganciclovir. The effect of the Ad-RSV-tk/ganciclovircombination on cell viability is then confirmed by the calorimetric testemploying MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide, according to the technique described by Mosman (J. Immunol.Meth. 65 (1983) 55), or more accurately by cell counting. Briefly,vascular smooth muscle cells (VSMC) are set up in culture by enzymaticdigestion of NZW rabbit aorta according to a method adapted from Chamleyet al. (Cell Tissue Res. 177: 503-522 1977). The cells are maintained inthe presence of 20% of foetal calf serum and used for all the tests (seebelow) before the tenth passage. In all our experiments, smooth musclecells are characterized by immunolabelling using anti-αSM-actin antibody(Sigma).

[0075] In order to measure the cytotoxic activity of theAd-RSV-TK/ganciclovir combination, rabbit aorta VSMC are incubated inthe presence of the adenovirus diluted in culture medium (DMEM, 0.5%FCS). After approximately one hour at 37° C. in a humid atmosphere, themedium containing the adenoviral solution is aspirated off and replacedby culture medium (DMEM, 0.5% FCS) for a period of 18 to 24 hours.Different concentrations of ganciclovir are then added in an FCS-rich(10%) medium. Four days after the addition of ganciclovir, the cells arecounted (100% cell viability corresponding to cells not transduced withAd-RSV-TK and not treated with ganciclovir).

[0076] The Ad-RSV-TK/ganciclovir combination induces a cytotoxic effectwith respect to rabbit VSMC (see FIG. 3). This cytotoxicity varies inaccordance with the ganciclovir concentration and the multiplicity ofAd-RSV-TK infection. Under our experimental conditions, i.e. four daysof incubation in the presence of 10% FCS, the Ad-RSV-TK (M.O.I.1000)/ganciclovir (25 μM) combination brings about complete cytolysis.Under these experimental conditions where a high multiplicity ofinfection enables the majority of the cells to be transduced, the IC₅₀is 0.3 μM. At a low multiplicity of infection (M.O.I. 10), the IC₅₀ isless than 5 μM. Thus, the concentrations of ganciclovir which are activein vitro on SMC are compatible with a therapeutic use. In effect, inpatients treated for viral infection with a non-toxic dose ofganciclovir, it is possible to reach plasma concentrations above 15 μM(Paul and Dummer, Am. J. Med. Sci. 4: 272-277, 1992).

[0077] In addition, this in vitro study demonstrates that it is possibleto induce a major cytotoxic effect in spite of a small percentage oftransduction by the Ad-RSV-TK adenovirus. The presence of the HSV-TKprotein was demonstrated by indirect immunofluorescence using monoclonalantibodies specific for HSV-TK (monoclonal antibody 4C8, YaleUniversity). In rabbit (and human) VSMC treated with Ad-RSV-TK, thelocalization of the TK protein is cytoplasmic but also nuclear. We haveshown in this way that the use of a multiplicity of infection of 10 isassociated with a transduction of fewer than 5% of the VSMC. Generallyspeaking, at an equivalent multiplicity of infection, the percentage ofcells transduced by Ad-RSV-TK is similar to that obtained using acontrol adenovirus coding for β-galactosidase (e.g. more than 90% ofcells transduced at a multiplicity of infection of 1000). These datahence demonstrate that the transduction of fewer than 5% of VSMC bringsabout a substantial cytotoxicity in the presence of an optimalconcentration of ganciclovir (see FIG. 3: 80% drop in cell viability at25 μM). Immunodetection of the HSV-TK protein hence illustrates themagnitude of the “bystander” effect observed on VSMC treated with theAd-RSV-TK/ganciclovir combination. This “bystander” effect may have itscounterpart in vivo. In particular, these data strongly suggest that alimited transfer of Ad-RSV-TK adenovirus, in particular into apathological artery, may lead to a significant reduction of theneointimal mass, which is rich in SMC and responsible for restenosis inthe patient.

[0078] Moreover, the cytolytic effect is selective, since neither simpletreatment with ganciclovir nor transduction with Ad-RSV-TK per se isassociated with cell death. The cytotoxicity of theAd-RSV-TK/ganciclovir combination has been confirmed by the calorimetrictest employing MTT. Lastly, similar results, namely a selective toxicityin the presence of ganciclovir and adenovirus, have been observed on aprimary culture of human smooth muscle cells.

[0079] Hence these data demonstrate the effective blocking of theproliferation of VSMC in vitro by Ad-RSV-TK.

Example 3 Arterial Transfer of a Recombinant Adenovirus via thePercutaneous Route

[0080] This example describes the development of an especially effectivetechnique for gene transfer via the percutaneous route. This techniqueis based on the use of a balloon catheter with hydrogel. The resultspresented show that, most advantageously, this technique enables certainfavoured cell populations to be infected effectively, in particular forthe treatment of restenosis.

[0081] This example was carried out using a defective recombinantadenovirus comprising the E. coli μ-galactosidase gene under the controlof the RSV RSV promoter and of a nuclear localization signal. Theconstruction of this adenovirus has been described, in particular, inStratford-Perricaudet et al., (J. Clin. Invest. 90 (1992) 626).

[0082] The experiments were carried out on New Zealand white rabbitsanaesthetized with acepromazine and maintained under pentobarbital. Genetransfer was performed at the external iliac artery.

[0083] The adenovirus Ad-RSV.β-Gal (1-2×10¹⁰ pfu in 100 μl of phosphatebuffer) was deposited on a balloon catheter previously coated withhydrogel (Hydroplus, Mansfield Medical, Boston Scientific Corp.,Watertown, Mass.) (Riessen et al., Hum. Gene Ther. 4 (1993) 749). Thecatheter used is a balloon catheter 2 cm in length and between 2.5 and 3mm in diameter. The catheter was then introduced, using a protectivesleeve, into the right femoral artery. A pressure of one atmosphere wasthen applied, and the catheter was thereafter directed into the externaliliac artery where a pressure of 6 atmospheres was then applied to theballoon for 30 minutes. This experiment was carried out on 27 rabbits. 3to 28 days after administration, the animals were sacrificed byoverdosing with pentobarbital.

[0084] Transfer of the Gene into the Arterial Wall and its ExpressionTherein

[0085] The arteries of the sacrificed animals were isolated, and theexpression of β-galactosidase was detected by staining in the presenceof X-gal according to the technique of Sanes et al., (EMBO J. 5 (1986)3133). For each animal, at least two arterial segments were eithermounted on OCT (Miles Laboratories Inc.; IL) for frozen-sectionexperiments, or coated with paraffin, cut into 6 μm sections andcounterstained with haematoxylin and eosin. Expression was consideredpositive only when a deep blue colouration was observed in the nucleus.The results obtained show clearly that the arteries of the infectedanimals display a blue colouration characteristic of μ-gal. Amicroscopic analysis discloses that there is no residual intactendothelium, but that the continuity of the internal elastic lamina ispreserved. Microscopic analysis also shows that the cells of the mediahave been infected with the adenoviruses and express the transferredgene. More precisely, whereas, in the case of an administration bydouble-balloon catheter, only 0.4% of the cells of the media have beeninfected, 9.6% are infected using a balloon catheter coated withhydrogel (see morphometric analysis below). Furthermore, the 9.6% arecalculated relative to the total thickness of the media but, in thesuperficial layers of the media, 100% of the cells are infected. Theseresults are far better than those obtained with double-ballooncatheters, or by transfer of naked genes or by means of liposomes. Theseexperiments demonstrate the extent to which adenoviruses can constitutean especially advantageous vector for the administration of suicidegenes for the purpose of treatment of restenosis.

[0086] MorPhometric Analysis

[0087] The efficacy of transfer was determined on 7 treated rabbits. Allthese animals received 5×10⁹ pfu of adenovirus to infect an arterialsegment 2 cm in length, so that the multiplicity of infection is similarfor each animal. For each iliac artery transfected, two serial segments5 mm in length were taken from the target zone and, for each segment, atleast three sections at random were examined under the light microscopeafter staining with X-gal. On each section, the efficacy of transfer wasdetermined from the ratio of stained media cells to the total number ofmedia cells. In all, more than 30×10³ cells originating from arteriesinfected with the adenoviruses (48 sections) were counted. The meanpercentage of infected media cells is 4.02%, with values which can reach9.6%. In the case of a transfer with a double-balloon cathether, themean percentage is only 0.18%.

[0088] Kinetics of Expression

[0089] To determine the duration of expression of the gene transferredby the adenoviruses according to the invention, a study of theexpression of β-gal was carried out over time on 20 rabbits treatedeither using a double-balloon catheter (10 animals) or using a ballooncatheter impregnated with hydrogel (10 animals). For each group, 2animals were sacrificed on days 3, 7, 14, 21 and 28. Expression wasdetected by macroscopic and microscopic examination of arteries stainedwith X-gal as described above. The results obtained show, for eachgroup, a stable expression for 14 days, followed by a drop at 21 days.No expression is detected at 28 days. The same kinetics could bedemonstrated in a pathological artery in the atheromatous rabbit model.These results confirm the transient effect of the vectors of theinvention, which is especially advantageous and suited to the treatmentof restenosis, in particular on the atheromatous walls.

[0090] Selectivity of the Transfer and Expression with Respect toArterial Walls

[0091] In order to check the possible dissemination to other tissues ofthe adenoviruses injected, in all the animals sacrificed 3 days afterinjection, tissue samples originating from the liver, brain, testicles,heart, lung, kidney and skeletal muscle, as well as an arterial segmentadjacent to the site treated, were removed immediately after sacrifice.On each sample, gene transfer and expression were demonstrated by PCR(by means of probes directed against the gene coding for adenovirusprotein IX and against the lacZ gene) and histochemistry. The resultsobtained show that none of the samples removed from the animals treatedwith a balloon catheter coated with hydrogel displays staining in thetissues tested. Similarly, no presence of virus could be detected by PCRin the samples tested, even using an optimized and very sensitiveprotocol of 45 amplification cycles.

[0092] These results demonstrate the efficacy of the mode ofadministration according to the invention for delivering therapeuticgenes very locally.

Example 4 Arterial Transfer of the Adenovirus Ad-RSV-TK

[0093] This example demonstrates the properties of the TK adenovirus ofthe invention for the treatment of restenosis by selective transfer intoan atheromatous artery.

[0094] Animal Model

[0095] The efficacy of the arterial transfer was evaluated in a model ofrestenosis in New Zealand white rabbits. The rabbits were subjectedbeforehand to a cholesterol-rich (1%) diet for two weeks. The iliacartery was abraded by five successive inflations using a latex balloon(4F). The animals were subjected again to ahypercholesterolaemia-inducing diet for six weeks. Arterial transfer wasperformed percutaneously according to the procedure described above, atthe damaged artery. The adenovirus Ad-RSV-TK (4×10⁹ pfu in 40 μl ofphosphate buffer) was accordingly deposited on a balloon catheterpreviously coated with hydrogel (Hydroplus, Mansfield Medical, BostonScientific Corp., Watertown, Mass.) (Riessen et al., Hum. Gene Ther. 4(1993) 749). The catheter used is a balloon catheter 2 cm in length and2.5 mm in diameter.

[0096] Based on a double lesion following abrasion and angioplasty, thismodel of restenosis, and not only of stenosis, enables the efficacy ofadenoviral transfer of a suicide gene into an atheromatous artery to beevaluated.

[0097] In the light of the in vitro experimental data, and in order toverify the selectivity of the treatment with Ad-RSV-TK, the animals weredivided into two groups, treated or otherwise with ganciclovir. Thetreatment with ganc-clovir was prolonged for five days (from the secondto the seventh day following angioplasty) at the rate of 2×25 mg/kg/day.

[0098] Morphometric Analysis

[0099] Six weeks after angioplasty, the animals were sacrificed withpentabarbital, and the iliac arteries were fixed and removed for thepurpose of morphometric analysis. The contours of the lumen as well asthose of the internal/external elastic limiting membranes were evaluatedafter staining with orcein/haematoxylin. In all, six blocks wereanalysed per artery, including 4 lying within the angioplasty zone andtwo immediately upstream and downstream of this zone. For each block,three adjacent sections were analysed. Briefly, different parameterssuch as luminal diameter and intima/media ratio were calculated. Samplesfor which these contours could not be revealed as a result of a ruptureof the internal elastic limiting membrane or of a thrombotic occlusionwere excluded.

[0100] Morphometric analysis reveals a high intima/media ratio in thecontrol group which has been subjected to adenoviral transfer byangioplasty but not treated with ganciclovir (5.73+/−0.81, n=3). Theseverity of the lesion hence enables the efficacy of a gene transfertreatment to be evaluated on a pathological artery. In addition, asshown by the immunohistochemical study, the lesions induced in thisanimal model are rich in macrophages, but also rich in smooth musclecells which constitute the therapeutic target of the gene transfer.These data collectively bring out the value of the model used, which isnot based on a simple endothelial abrasion in a healthy artery, andconsequently mimics at least partially the pathology of postangioplastyrestenosis in man.

[0101] Morphometric analysis shows that the intima/media ratio isreduced by 42% (p<0.05) in the group of animals subjected to theAd-RSV-TK/ganciclovir combination (3.30 +/−1.26, n=6). The significantreduction in this parameter demonstrates that local transfer of the TKgene by a recombinant adenovirus, in combination with the administrationof ganciclovir, constitutes a promising therapeutic approach as apreventive treatment for postangioplasty restenosis.

1. Use of a defective recombinant adenovirus containing a suicide genefor the preparation of a pharmaceutical composition intended for thetreatment of restenosis.
 2. Use of a defective recombinant adenoviruscontaining a suicide gene for the preparation of a pharmaceuticalcomposition intended for the treatment of restenosis by selectivetransfer of the said gene into the smooth muscle cells of theatheromatous plaque.
 3. Use according to claim 1 or 2, characterized inthat the suicide gene is chosen from the thymidine kinase gene and thecytosine deaminase gene.
 4. Use according to claim 1 or 2, characterizedin that the suicide gene is the human herpesvirus thymidine kinase(HSV-1 TK) gene.
 5. Use according to one of the preceding claims,characterized in that the suicide gene is placed under the control of apromoter permitting its expression in infected cells.
 6. Use accordingto claim 5, characterized in that the promoter is chosen from viralpromoters, preferably the RSV LTR and CMV promoter.
 7. Use according toone of the preceding claims, characterized in that the adenoviruscomprises the ITRs, a sequence permitting encapsidation and the suicidegene.
 8. Use according to claim 7, characterized in that the adenoviruscomprises the ITRs, a sequence permitting encapsidation and the suicidegene, and in which the E1 gene and at least one of the genes E2, E4,L1-L5 is non-functional.
 9. Use according to claim 8, characterized inthat the adenovirus comprises the ITRs, a sequence permittingencapsidation and the suicide gene, and in which the E1 gene and the E4gene is rendered nonfunctional.
 10. Use according to claim 9,characterized in that the adenovirus comprises the ITRs, a sequencepermitting encapsidation and the suicide gene, and in which all or partof the E1 and E4 regions are deleted.
 11. Use according to one of thepreceding claims, characterized in that the adenovirus is an adenovirusof human origin, preferably chosen from the serotypes Ad2 and Ad5. 12.Use according to one of claims 1 to 10, characterized in that theadenovirus is an adenovirus of animal origin, preferably chosen fromcanine adenoviruses.
 13. Use according to one of claims 1 to 12,characterized in that the adenovirus is impregnated in a hydrogel. 14.Use according to claim 13, characterized in that the hydrogel isdeposited on a balloon catheter.
 15. Use according to claim 11,characterized in that the adenovirus is administered via a ballooncatheter of the perfusion catheter type.
 16. Use according to claim 15,characterized in that the adenovirus is administered via a catheter ofthe channelled balloon catheter type.
 17. Use according to claim 15,characterized in that the adenovirus perfused via a catheter of theperfusion balloon catheter type is impregnated in a hydrogel.
 18. Useaccording to one of claims 1 to 12, characterized in that the adenovirusis impregnated in poloxamer.
 19. Use according to claim 15,characterized in that the adenovirus perfused via a catheter of theperfusion balloon catheter type is impregnated in poloxamer. 20.Pharmaceutical composition comprising a defective recombinant adenovirusimpregnated in a hydrogel.
 21. Pharmaceutical composition according toclaim 20, characterized in that the defective recombinant adenoviruscontains a suicide gene.
 22. Device for the percutaneous administrationof genes, characterized in that it comprises a balloon catheter coatedwith a hydrogel, the hydrogel being impregnated with a defectiverecombinant adenovirus containing the said gene.
 23. Device according toclaim 22, characterized in that the administration of genes is carriedout selectively at the atheromatous plaque.
 24. Device according toclaim 23, characterized in that the administration of genes is carriedout selectively at the smooth muscle cells.
 25. Device according toclaim 24, characterized in that, when genes are administered, thisadministration takes place with a selectivity of greater than 95%. 26.Device according to claims 23 to 25, characterized in that theadministration of genes is followed by a treatment with ganciclovir. 27.Device according to claim 26, characterized in that the percentage ofinfected cells is greater than or equal to 0.2%.
 28. Method oftherapeutic treatment of restenosis, characterized in that it comprisesthe percutaneous administration of genes by means of a balloon cathetercoated with a hydrogel, the hydrogel being impregnated with a defectiverecombinant adenovirus containing the said gene.
 29. Method oftherapeutic treatment of restenosis according to claim 28, characterizedin that the administration of genes takes place selectively at theatheromatous plaque.
 30. Method of therapeutic treatment of restenosisaccording to claim 29, characterized in that the administration of genestakes place selectively at the smooth muscle cells.
 31. Method oftherapeutic treatment of restenosis according to claim 30, characterizedin that the administration of genes takes place with a selectivity ofgreater than 95%.
 32. Method of therapeutic treatment of restenosisaccording to claim 31, characterized in that the administration of TKsuicide genes is followed by a treatment with ganciclovir.
 33. Method oftherapeutic treatment of restenosis according to claim 32, characterizedin that it induces a “bystander” effect.
 34. Method of therapeutictreatment of restenosis according to claim 33, characterized in thatthis induced bystander effect permits a therapeutic efficacy even with asmall percentage of infected cells.
 35. Method of therapeutic treatmentof restenosis according to claim 34, characterized in that thepercentage of infected cells is greater than or equal to 0.02%.